Identification and expression of acid beta-glucosidase mutations causing severe type 1 and neurologic type 2 Gaucher disease in non-Jewish patients.

Abstract

Gaucher disease, the most prevalent lysosomal storage disease, occurs in three subtypes, all resulting from mutations in the acid ␤ -glucosidase gene. Molecular studies in five severely affected type 1 and two type 2 Gaucher disease patients of non-Jewish descent identified six new mutations: K74X, W179X, G195E, S271N, V352L, and a two-base deletion in exon 10 (1450del2). Two additional mutations identified in these patients (R48W and G202R) have been reported previously, but were not expressed or characterized. Heterologous expression in Sf9 cells using the baculovirus system revealed that the missense mutations, R48W and V352L, had 14 and 7%, respectively, of the specific activity based on cross-reacting immunologic material expressed by the normal allele. In contrast, the G195E, G202R, and S271N mutant alleles were more severely compromised with only 1-2% of the normal expressed specific activity based on cross-reacting immunologic material. Structural distortion at the active site was probed by comparing the interaction of the mutant enzymes with active site-directed inhibitors (castanospermine, conduritol B epoxide and deoxynojirimycin). R48W, G202R, and S271N were normally inhibited, whereas the V352L and G195E mutant enzymes had significantly decreased binding affinity. These mutations further expand the genetic heterogeneity in the lesions causing Gaucher disease types 1 and 2, and further delineate genotype/phenotype correlations and functional domains within the acid ␤ -glucosidase gene. ( J. Clin. Invest. 1997. 99:2530-2537.)

Gaucher disease: expression and characterization of mild and severe acid β-glucosidase mutations in Portuguese type 1 patients

“…As shown in Figure 1, lane 10, the normal human acid -glucosidase cDNA produced a multi-band pattern that strongly reacted with polyclonal anti-human acid -glucosidase antibodies. Consistent with earlier studies, 52 treatment with N-Glycanase™ demonstrated that the multiple bands with molecular weights ranging from 63 kDa to about 56 kDa represented differentially glycosylated forms of the enzyme (data not shown). Similar multi-band patterns were seen for all of the expressed mutant cDNAs.…”

“…51,52 The complete sequence of each mutagenized cDNA was determined to confirm that no spurious mutations were incorporated during the mutagenesis procedure. The mutant cDNAs were then cloned into the EcoRI site of the baculovirus expression vector, pVL1392.…”

“…53 Pure recombinant baculovirus clones containing the normal or mutant cDNA for acid -glucosidase were isolated by plaque hybridization, amplified, titered, and used at a multiplicity of infection greater than 10 to infect Sf9 cells. 53 The previously characterized common mutations, N370S and L444P, 51,52,54,55 were re-expressed for comparative purposes.…”

“…Specific activities based on the amount of cross-reacting immunological material, CRIM specific activity (CRIM SA), were determined as described. 51,52,54 The relative amounts of CRIM per unit of glucosylceramide-cleaving activity for each mutant allele was determined by computer analysis of the immunoblotting membrane using the NIH Image™ program, version 1.60, and referenced to that of the normal enzyme. .…”

“…For comparison, the previously studied N370S and L444P alleles were re-expressed. 51,52,54 Using the fluorescently-labeled natural substrate, NBD-GC, the expressed normal acid -glucosidase allele had approximately 9.6 ϫ 10 -2 µmol/min/mg (U/mg) of glucosylceramide-cleaving activity in the cell lysates. No activity towards NBD-GC was detected in Sf9 cells infected with the recombinant virus containing the antisense acid -glucosidase construct.…”